The invention was supported, in whole or in part, by funding from the Government. The Government has certain rights in the invention.
Fungal species are the commercial source of many medicinally useful products, such as antibiotics (e.g., beta-lactam antibiotics such as penicillin, cephalosporin, and their derivatives), anti-hypercholesterolemic agents (e.g., lovastatin and compactin), immunosuppressives (e.g., cyclosporin), and antifungal drugs (e.g., pneumocandin and echinocandin). All of these drugs are fungal secondary metabolites, small secreted molecules that fungi utilize against competitors in their microbial environment. Fungi also produce commercially important enzymes (e.g., cellulases, proteases, and lipases) and other products (e.g., citric acid, gibberellic acid, natural pigments, and flavorings).
The production of secondary metabolites, enzymes, and other products is regulated by coordinated gene expression. For example, the production of penicillin is limited by the activity of two enzymes, encoded by the ipnA and acvA genes. PacC, a zinc-finger transcription factor, binds to sequences upstream of these two genes. Moreover, increased activity of PacC leads to both increased enzyme activity and penicillin production.
Our understanding of transcriptional regulation of secondary metabolite production, as exemplified above, has increased greatly over the past decade. To date, however, the use of genetically-engineered transcription factors has not been applied to increase production of commercially-important fungal products. In contrast, methods to increase production of penicillin currently rely upon mutagenesis and selection for mutants which display increased secondary metabolite production.
The invention provides a means to increase the production of secondary metabolites in fungi by genetic manipulation of the fungal organism itself. The ability to increase fungal secondary metabolite production has at least two important applications. First, it will allow increased production of existing secondary metabolites which are useful in clinical and experimental settings. Second, increasing production of secondary metabolites will facilitate identification of new compounds in fungi that otherwise make undetectable levels of these compounds in the laboratory.
Accordingly, in one aspect, the invention features a two-part chimeric transcription factor including (i) a pre-activated transcription factor functional in a fungal strain, and (ii) a transcription activation domain that is different from the transcription activation domain naturally associated with the transcription factor. In a preferred embodiment, the transcriptional activity of the chimeric transcription factor is greater than the transcriptional activity naturally associated with the pre-activated transcription factor. In another preferred embodiment, the pre-activated transcription factor is pre-activated by truncation. In a related preferred embodiment, the pre-activated transcription factor includes a substitution of a serine or threonine residue with an alanine, aspartic acid, or glutamic acid residue, wherein the substitution pre-activates the transcription factor (e.g., by mimicking or otherwise altering phosphorylation). In another preferred embodiment, the transcription factor is a member of the PacC family (defined below) and can be pre-activated. In a related preferred embodiment, the pre-activated transcription factor contains portions of the amino acid sequence shown in FIG. 1 (SEQ ID NOs: 1-6).
In another aspect, the invention features a vector including DNA encoding a chimeric transcription factor including (i) a pre-activated transcription factor functional in a fungal strain, and (ii) a transcription activation domain that is different from the transcription activation domain naturally associated with the transcription factor. The DNA is operably linked to a promoter capable of directing and regulating expression of the chimeric transcription factor in a fungal strain.
The transcription factor encoded within the vector described above is expressed in a fungal cell, such as a filamentous fungal cell, which produces the secondary metabolite of interest and in which expression of the transcription factor increases the production of the secondary metabolite by the cell. The secondary metabolite can be non-proteinaceous or it can be a protein or peptide.
In another aspect, the invention features a method of producing a secondary metabolite of interest, including the steps of (i) introducing into a fungal cell, such as a filamentous fungal cell, a vector including a promoter capable of controlling gene expression in the fungal cell, and a nucleic acid encoding a two-part transcription factor including a DNA-binding domain and a transcription activation domain; and (ii) culturing the fungal cell under secondary metabolite-producing conditions. In a preferred embodiment, the transcription activation domain is different from the transcription activation domain naturally associated with the DNA-binding domain. In other preferred embodiments, the transcription factor is a pre-activated transcription factor (pre-activated by substitution of a serine or threonine residue with an alanine, aspartic acid, or glutamic acid residue, or pre-activated by truncation). In other preferred embodiments, the DNA binding domain of the transcription factor is from a fungal transcriptional activator or from a fungal transcriptional repressor.
By xe2x80x9cpre-activated transcription factorxe2x80x9d is meant a transcription factor or fragment thereof that, compared to the precursor molecule, is capable of 1) increased binding, either direct or indirect, to a specific DNA sequence located in a gene regulatory region (e.g., a promoter), or 2) increased transcription activating properties. Pre-activated transcription factors may be able to activate transcription from promoters, but this is not necessarily the case. For example, a transcription factor DNA-binding domain with binding properties but no transactivation activity is considered to be a pre-activated transcription factor. xe2x80x9cPre-activation by truncationxe2x80x9d or xe2x80x9cpre-activated by truncationxe2x80x9d means that removal of a portion of the protein leads to pre-activation. This occurs in vivo through proteolytic cleavage. In the invention, pre-activation by truncation is achieved with the use of DNA that encodes a pre-activated form of the protein, excluding portions of the protein that would be proteolytically cleaved in vivo.
By xe2x80x9csubstantially identicalxe2x80x9d is meant a polypeptide or nucleic acid exhibiting at least 50%, preferably 85%, more preferably 90%, and most preferably 95% identity to a reference amino acid or nucleic acid sequence. For polypeptides, the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids. For nucleic acids, the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides.
By xe2x80x9cpromoterxe2x80x9d is meant a sequence sufficient to direct and/or regulate transcription. Also included in the invention are those elements which are sufficient to render promoter-dependent gene expression controllable for cell type-specific, tissue-specific, temporal-specific, or inducible by external signals or agents; such elements may be located in the 5xe2x80x2 or 3xe2x80x2 or intron sequence regions of the native gene.
By xe2x80x9coperably linkedxe2x80x9d is meant that a gene and one or more regulatory sequences are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequences.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.